The present invention relates to recombinant Factor C (rFC) of a horseshoe crab, produced in an insect cell system. The invention also relates to vectors for producing the protein by recombinant DNA methods and to methods for using the recombinant Factor C to detect endotoxins in a sample or for removal of endotoxins from a sample by affinity methods.
The amoebocytes of horseshoe crabs contain an efficient coagulation cascade system which is activated by endotoxin, also known as lipopolysaccharide (LPS) from Gram negative bacteria. The enzymatic components of the coagulation cascade and the molecular events responsible for the subsequent gelation of the amoebocyte lysate have been characterized in Tachypleus tridentatus1 and Carcinoscorpius rotundicauda2,3,4. Factor C has been shown to be the intracellular endotoxin-sensitive serine protease that initiates the coagulation cascade system5.
By spiking, the Limulus amoebocyte lysate (LAL) test detects femtogram levels of LPS6. Owing to its extreme sensitivity, the amoebocyte lysate, in particular, the LAL has been developed into a commercial assay for widespread use in the detection of pyrogenic LPS in drugs and other pharmaceutical products7,8. This assay is based on the LPS-induced coagulation reaction of the lysate, culminating in formation of a gel clot. However, (a) the possible lack of specificity due to 1-3 xcex2-D glucan and (b) the batch-to-batch variation in the sensitivity of commercial lysate to LPS, due to seasonal and geographical differences in the starting material9 has prompted our laboratory to employ recombinant DNA technology to genetically-engineer Factor C as an alternative source of novel xe2x80x9climulus lysatexe2x80x9d for endotoxin detection.
cDNAs encoding Factor C have been cloned1,10,15. There are six potential glycosylation sites in the amino acid sequence of the Factor C from Carcinoscorpius rotundicauda (CrFC)10,15. Cloned cDNA encoding CrFC has been expressed in E. coli11 and also in yeast expression systems12, 24. The rFC obtained from yeast was found to be immunoreactive and capable of binding LPS, although only limited amounts of rFC produced in yeast were soluble13,14. Also, it was found that LPS could not activate the enzymatic activity of yeast rFC, thus, a direct enzyme-based LPS detection is not possible using rFC produced in yeast14.
Since the early 1970s, the diagnostic potential of LAL for endotoxemia has been recognized to be extremely important for timely and effective treatment. Thus, many studies have been conducted to improve the sensitivity of endotoxin assays by changing the formulation of the LAL and assay methodology25. Since blood (plasma) components interfere with the test, various methods to remove inhibition and/or enhancement have been developed. Furthermore, the LAL gelation clot method had been criticized as being subjective, semiquantitative and prone to variations in interpretations by different workers. Thus, advent of an improved fluorimetric assay for using LAL in LPS detection of plasma would be desirable. Even more desirable would be the compatibility of this assay for both LAL and a recombinant Factor C (rFC) in their sensitivity to LPS. Furthermore, this demonstrates the possible diagnostic utility of rFC for endotoxemia.
The present inventors believed that expression in insect cells rather than in a prokaryotic or simple eukaryotic expression system is suitable for producing rFC with full biological activity. Furthermore, horseshoe crabs and insects belong to the same phylum, Arthropoda, and so insect cells might more closely resemble the cells of the horseshoe crab than yeast cells in their physiology and biochemistry. Thus, rFC produced in insect cells might more closely resemble the protein as purified from the horseshoe crab and retain the bioactivity of having a serine protease activity activated by LPS.
The present invention relates to genetic engineering of a bioactive rFC, which unequivocally exhibits full biological functionality. It is capable of specifically recognizing and binding LPS and lipid A in both free and immobilized forms. Interference from 1-3 xcex2-D-glucan, which switches on the alternate pathway in the coagulation cascade in conventional LAL, is not anticipated in assays of the present invention that use only Factor C as the LPS-binding, serine protease enzyme. Both the LPS-activated enzymatic assays of rFC and the enzyme linked immunosorbent assay (ELISA) lipid A binding assay could be formulated into a rapid high throughput mass screening test for LPS. Thus, a novel generation of xe2x80x9climulus amoebocyte lysatexe2x80x9d has been invented, being capable of rapid and sensitive diagnosis and removal of subpicogram levels of endotoxin. The invention provides a standardized and convenient source of enzyme-based diagnostic reagent for detection of the ubiquitously contaminating endotoxin in pharmaceutical products. This inexhaustible supply of genetically-engineered Factor C can be easily standardized to circumvent batch-to-batch variations in sensitivity to LPS, a problem faced by the conventional LAL industry. Furthermore, the ability of the rFC of the invention to protect mice from endotoxemia, as well as its bacteriostatic activity, adds to its value in in vivo applications. Furthermore, the availability of rFC obviates the need for routine harvesting of the horseshoe crab for procurement of their amoebocyte lysate, and therefore, conserves this endangered xe2x80x9cliving fossilxe2x80x9d.
The present inventors have succeeded in expressing biologically active rFC using recombinant baculoviruses and other vectors appropriate for expression of heterologous DNA in insect host cells. The rFC obtained is enzymatically active. Thus, expression of rFC in insect cells is a convenient and economical source of rFC protein for use in rapid, sensitive, specific and quantitative determination of LPS in pharmaceutical products and other biological fluids.
Thus, the present invention comprises purified rFC that is enzymatically active. The phrase xe2x80x9cenzymatically activexe2x80x9d means that the Factor C protein has the biological activity of binding LPS or lipid A, being activated as to its serine protease activity upon LPS or lipid A binding. Enzymatically active rFC will induce coagulation of an amoebocyte lysate and will also cleave synthetic substrates such as, but not limited to, Boc-Val-Pro-Arg-MCA, Mu-Val-Pro-Arg-AFC and Boc-Val-Pro-Arg-pNA.
The present invention is also embodied in a method for producing substantially purified, enzymatically active rFC. The method comprises expressing DNA encoding a Factor C protein having the enzymatic activity described above in a culture of insect cells, then isolating the enzymatically active Factor C protein. The isolation preferably includes an ultrafiltration step. The purification preferably also includes a step of gel-filtration chromatography on a matrix having an exclusion limit of 100 kilodaltons. The gel filtration is preferably applied after the ultrafiltration. The exclusion limit of the gel filtration matrix can vary substantially; an effective matrix will provide at least about a 4-fold increase in the serine protease activity of an ultrafiltered crude preparation as measured by the fluorometric assay described herein.
The present invention also encompasses host-vector systems for expressing enzymatically active rFC. The host cells in these embodiments of the invention are insect cells, preferably leptidopteran cells. The vectors in these embodiments support replication of inserted DNA in insect cells and expression of heterologous DNA in insect cells. The vectors are preferably baculovirus or plasmid vectors. The heterologous DNA is sufficient to encode a Factor C enzyme of a horseshoe crab, preferably of the genus Carcinoscorpius, Tachypleus or Limulus. The recombinant Factor C will preferably at least have the LPS binding activity of Factor C and more preferably will have both LPS binding activity and serine protease activity. Preferred heterologous DNA is a polynucleotide having the sequence shown in SEQ ID NO:1 or SEQ ID NO:3.
The present invention is also embodied in assays for endotoxin comprising contacting a sample to be assayed for the presence of endotoxin or LPS or Lipid A with enzymatically active rFC according to the invention and measuring the serine protease activity of the rFC. The amount of serine protease activity of the rFC will reflect its activation due to binding of LPS or Lipid A or of another endotoxin known in the art to bind to Factor C of a horseshoe crab. The serine protease activity is conveniently measured by any method known in the art but is preferably measured by a chromogenic or fluorogenic method. In such a method formation of a product from a substrate by cleavage of the substrate by the serine protease activity of the rFC, resulting in a change in color or in fluorescence emission, is measured. Preferred substrates for such a chromogenic or fluorogenic assay are N-t-BOC-Val-Pro-Arg-MCA, Mu-Val-Pro-Arg-AFC and Boc-Val-Pro-Arg-pNA.
Additional embodiments of the invention include immunologic methods for assaying the presence of Lipid A or LPS or endotoxin in a sample. These methods of the invention rely upon binding of antibody that specifically binds to Factor C and subsequent detection or quantitation of the amount of the Factor C-antibody complex. In a preferred embodiment, the sample to be assayed is contacted with immobilized antibody that specifically binds to Lipid A or LPS or endotoxin as the ligand to form immobilized ligand. The immobilized ligand is then contacted with rFC according to the present invention to form immobilized rFC. Then the immobilized rFC is contacted with a second antibody that specifically binds the rFC. Finally, the presence or preferably the amount of the rFC-second antibody complex is determined. This determination can be performed by any method typical in the art such as a third antibody that binds the second antibody, perhaps through its Fc portion, or the like. In an alternate embodiment of this aspect of the invention, the second antibody is omitted and the enzymatic activity of the immobilized rFC is measured.
In another embodiment of the invention, the specific binding of LPS or lipid A to rFC is employed in a BIACORE(trademark) assay (Pharmacia Biotech). By immobilizing the rFC on the substrate plate of the BIACORE(trademark) apparatus, the presence of LPS or lipid A in a sample can be detected. Optimization of the amount of the rFC to be immobilized for a given load of LPS in a sample is considered within the skill of the ordinary practitioner. The BIACORE(trademark) apparatus is operated in accord with the manufacturer""s instructions.
Also, the present invention is embodied in methods for removal of endotoxin from a sample, wherein immobilized rFC is contacted with the sample, under conditions such that endotoxin in the sample binds the immobilized rFC, then the bound endotoxin is separated from the sample.